U.S. patent number 6,147,060 [Application Number 08/840,706] was granted by the patent office on 2000-11-14 for treatment of carcinomas using squalamine in combination with other anti-cancer agents.
This patent grant is currently assigned to Magainin Pharmaceuticals. Invention is credited to Jon Williams, Michael Zasloff.
United States Patent |
6,147,060 |
Zasloff , et al. |
November 14, 2000 |
Treatment of carcinomas using squalamine in combination with other
anti-cancer agents
Abstract
A method for treating a tumor includes a first treatment
procedure using a conventional cancer treatment technique, and a
second treatment procedure which includes administering an
effective amount of squalamine. The first treatment procedure may
be a treatment with one or more conventional cytotoxic chemical
compounds. As examples, the cytotoxic chemical compound may be a
nitrosourea (such as BCNU), cyclophosphamide, adriamycin,
5-fluorouracil, paclitaxel and its derivatives, cisplatin or other
platinum containing cancer treating agents. The cytotoxic chemical
compound and the squalamine may be administered by any suitable
route. The first treatment procedure may take place prior to the
second treatment procedure, after the second treatment procedure,
or the two treatment procedures may take place simultaneously. In
one example, the first treatment procedure (e.g., a one time
intravenous dosage of BCNU) is completed before the second
treatment procedure with squalamine begins. As an alternative, the
first treatment procedure may be a conventional radiation treatment
regimen.
Inventors: |
Zasloff; Michael (Merion
Station, PA), Williams; Jon (Robbinsville, NJ) |
Assignee: |
Magainin Pharmaceuticals
(Plymouth Meeting, PA)
|
Family
ID: |
21776862 |
Appl.
No.: |
08/840,706 |
Filed: |
April 25, 1997 |
Current U.S.
Class: |
514/110; 424/649;
514/171; 514/34; 514/589 |
Current CPC
Class: |
A61K
31/575 (20130101); A61K 33/243 (20190101); A61P
35/00 (20180101); A61K 33/24 (20130101); A61K
2300/00 (20130101); A61K 33/24 (20130101); A61K
31/575 (20130101); A61K 31/575 (20130101); A61K
31/00 (20130101); A61K 31/575 (20130101); A61K
2300/00 (20130101) |
Current International
Class: |
A61K
31/575 (20060101); A61K 33/24 (20060101); A61K
031/66 (); A61K 031/56 (); A61K 031/70 (); A61K
031/175 (); A61K 033/24 () |
Field of
Search: |
;514/171,34,110,589
;424/649 |
References Cited
[Referenced By]
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|
Primary Examiner: Goldberg; Jerome D.
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Parent Case Text
This application claims priority to U.S. Provisional Application
No. 60/016,387, filed Apr. 26, 1996.
Claims
We claim:
1. A method for treating a tumor that is sensitive to a synergistic
combination of a cytotoxic chemical compound and squalamine,
comprising the step of: administering a synergistically effective
amount of at least one cytotoxic chemical compound in a first
treatment procedure; and administering a synergistically effective
amount of squalamine in a second treatment procedure.
2. A method according to claim 1, wherein the cytotoxic chemical
compound is a member selected from the group consisting of: a
nitrosourea, cyclophosphamide, adriamycin, 5-fluorouracil,
paclitaxel and its derivatives, cisplatin, methotrexate, thiotepa,
mitoxantrone, vincristine, vinblastine, etoposide, ifosfamide,
bleomycin, procarbazine, chlorambucil, fludarabine, mitomycin C,
vinorelbine, and gemcitabine.
3. A method according to claim 1, wherein the cytotoxic chemical
compound is BCNU.
4. A method according to claim 1, wherein the cytotoxic chemical
compound is cyclophosphamide.
5. A method according to claim 1, wherein the cytotoxic chemical
compound is cisplatin.
6. A method according to claim 1, wherein in the first treatment
procedure, the cytotoxic chemical compound is administered
intravenously.
7. A method according to claim 6, wherein in the second treatment
procedure, the squalamine is administered subcutaneously.
8. A method according to claim 6, wherein the first treatment
procedure takes place prior to the second treatment procedure.
9. A method according to claim 1, wherein the first treatment
procedure is completed before the second treatment procedure
begins.
10. A method according to claim 1, wherein the first treatment
procedure is a one time injection of the cytotoxic chemical
compound.
11. A method according to claim 10, wherein the cytotoxic chemical
compound is BCNU.
12. A method according to claim 10, wherein the cytotoxic chemical
compound is cyclophosphamide.
13. A method according to claim 10, wherein the cytotoxic chemical
compound is cisplatin.
14. A method according to claim 11, wherein in the second treatment
procedure, the squalamine is administered subcutaneously.
15. A method according to claim 1, wherein in the second treatment
procedure, the squalamine is administered orally.
16. A method according to claim 1, wherein in the second treatment
procedure, the squalamine is administered intravenously.
17. A method according to claim 1, wherein in the second treatment
procedure, the squalamine is administered subcutaneously.
18. A method according to claim 1, wherein the tumor is a CNS
tumor.
19. A method according to claim 1, wherein the tumor is a breast
tumor.
20. A method according to claim 1, wherein the tumor is a lung
tumor.
Description
BACKGROUND OF THE INVENTION
I. Information Relating to Previous Squalamine Applications
This invention relates to various methods for using squalamine.
Squalamine, having the structure illustrated in FIG. 1, is an
aminosterol which has been isolated from the liver of the dogfish
shark, Squalus acanthias. This aminosterol is the subject of U.S.
Pat. No. 5,192,756 to Zasloff, et al., which patent is entirely
incorporated herein by reference. Methods for synthesizing
squalamine have been devised, such as the methods described in WO
94/19366 (published Sep. 1, 1994). This PCT publication is entirely
incorporated herein by reference. This PCT application also relates
to U.S. patent application Ser. No. 08/023,347 (filed Feb. 26,
1993), which application also is entirely incorporated herein by
reference. Additional methods for synthesizing squalamine also are
described in U.S. Provisional Patent Appln. No. 60/032,378 filed
Dec. 6, 1996, which application also is entirely incorporated
herein by reference.
U.S. patent application Nos. 08/416,883 (filed Apr. 20, 1995) and
08/478,763 (filed Jun. 7, 1995) describe the use of squalamine as
an antiangiogenic agent. These U.S. patent applications are
entirely incorporated herein by reference. Additional uses of
squalamine (e.g., as a sodium/proton exchanger (isoform 3), or
NHE3, inhibiting agent and as an agent for inhibiting the growth of
endothelial cells) and squalamine synthesis techniques are
disclosed in U.S. patent application No. 08/474,799 (filed Jun. 7,
1995). This U.S. patent application also is entirely incorporated
herein by reference.
II. Information Relating to This Invention
About 50,000 new cases of CNS (central nervous system) tumors are
diagnosed each year. Of these, about 35,000 are metastatic tumors
(e.g., lung, breast, melanomas) and about 15,000 are primary tumors
(mostly astrocytomas). Astrocytomas, along with other malignant
gliomas (i.e., cancers of the brain), are the third leading cause
of death from cancer in persons between the ages of 15 and 34.
Treatment options for a patient with a CNS tumor are very limited.
Currently, surgery is the treatment of choice. Surgery provides a
definite diagnosis, relieves the mass bulkiness of the tumor, and
extends survival of the patient. The only post-surgery adjuvant
treatment which is known to work on CNS tumors is radiation, and it
can prolong survival. Radiation treatment, however, has many
undesirable side effects. It can damage the normal tissue of the
patient, including the brain tissue. Radiation also can cause the
patient to be sick (e.g., nausea) and/or to temporarily lose their
hair.
The other common post-surgery adjuvant cancer treatment,
chemotherapy, is relatively ineffective against CNS tumors.
Specifically, chemotherapy against CNS tumors with nitrosoureas is
not curative. Many other cancer treating agents have been studied
and tested, but generally they have a minimal effect on extending
survival.
In view of these limited treatment options, the current prognosis
for persons with CNS tumors is not good. The median survival term
for patients with malignant astrocytomas having surgery and no
adjuvant treatment is about 14 weeks. Radiation therapy after
surgery extends the median to about 36 weeks. The current two year
survival rate for all forms of treatment is less than 10%.
To maximize survival, it is critical to begin treatment in the
early stages of CNS tumor development. Typically, the extent of
tumor angiogenesis (i.e., blood vessel formation) correlates with
survival in the patient. CNS tumors are among the most angiogenic
of all human tumors. When the tumor is small, however, it is in an
"avascular" phase, and its growth is restricted by a diffusion
mechanism (i.e., the cells receive their nutrition, etc. by
diffusion into the cell). In this phase, the tumor is viable, but
not growing, and it is unable to spread. Over time, however,
angiogenesis begins and the tumor converts to a "vascular" phase.
In this phase, perfusion replaces diffusion as the growth
mechanism, and tumor growth is exponential (i.e., the tumor has its
own blood vessels to provide nutrients, etc.). Mitotic cells
cluster around new blood vessels and metastases occur in the
vascular phase (i.e., the tumor can spread to other areas in the
body). Therefore, by treating the tumor early (before it reaches
the vascular phase), one can hope to inhibit metastatic spread as
well as control the primary tumor.
Other types of cancer also are difficult to combat by known cancer
treatments. Lung cancer kills more Americans annually than the next
four most frequently diagnosed neoplasms combined. Estimates for
1994 indicate more than 170,000 new cases of lung cancer and
approximately 150,000 deaths (Boring et al.; CA Cancer J. Clin.
1994, 44: 7-26). Approximately 80% of primary lung tumors are of
the non-small cell variety, which includes squamous cell and large
cell carcinomas, as well as adenocarcinomas.
Single-modality therapy is considered appropriate for most cases of
early and late stage non-small cell lung cancer (NSCLC). Early
stage tumors are potentially curable with surgery, chemotherapy, or
radiotherapy, and late stage patients usually receive chemotherapy
or best supportive care. Intermediate stage or locally advanced
NSCLC, which comprises 25% to 30% of all cases of NSCLC, is more
typically treated with multimodality therapy. This is a stage of
tumor development when angiogenesis is a very important factor. New
blood vessels are needed to support further tumor growth and for
the development of metastases. Therefore, this stage is amenable to
treatment with antiangiogenic agents to prevent the development of
new blood vessels. The efficacy of this therapy can be further
improved by the combination of the antiangiogenic therapy with
cytotoxic chemotherapy or radiation therapy to eliminate existing
tumor.
Breast cancer also presents treatment difficulties using known
agents. The incidence of breast cancer in the United States has
been rising at a rate of about 2%/year since 1980, and the American
Cancer Society estimated that 182,000 cases of invasive breast
cancer were diagnosed in 1995. Breast cancer is usually treated
with surgery, radiotherapy, chemotherapy, hormone therapy, or
combinations of the various methods. Like other solid tumors,
breast cancer requires the development of new blood vessels to
support its growth beyond a certain size, and at that stage in its
development, it will be amenable to treatment with antiangiogenic
agents.
A major reason for the failure of cancer chemotherapy in breast
cancer is the development of resistance to the cytotoxic drugs.
Combination therapy using drugs with different mechanisms of action
is an accepted method of treatment which prevents development of
resistance by the treated tumor. Antiangiogenic agents are
particularly useful in combination therapy because they are not
likely to cause resistance development since they do not act on the
tumor, but on normal host tissue.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a method for treating
malignant and cancerous tumors using squalamine, in combination
with other, conventional cancer treating agents. In one aspect of
the invention, CNS tumors are treated; in another aspect, lung
tumors are treated; and in yet another, breast tumors are
treated.
In one method according to the invention, squalamine is used in
combination with conventional cancer treatments to treat tumors.
The tumor is treated by administering an effective amount of a
cytotoxic chemical compound in a first treatment procedure, and an
effective amount of squalamine is administered in a second
treatment procedure.
In this method, the cytotoxic chemical compound used in the first
treatment procedure is a conventional cancer treating agent.
Preferable agents include a nitrosourea, cyclophosphamide,
adriamycin, 5-fluorouracil, paclitaxel and its derivatives, and
cisplatin and related platinum compounds. These conventional cancer
treating agents are well known to those skilled in this art. Note,
M. C. Wiemann and Paul Calabresi, "Pharmacology of Antineoplastic
Agents," Medical Oncology, Chapter 10, edited by Paul Calabresi,
et. al., McMillan Publishing (1985). Medical Oncology is entirely
incorporated herein by reference. One particularly preferred
nitrosourea is BCNU, which also is known as carmustine. Another
preferred cytotoxic agent is cisplatin, and yet another is
cyclophosphamide. Other conventional cytotoxic chemical compounds,
such as those disclosed in Medical Oncology, supra., can be used
without departing from the invention.
The cytotoxic chemical compound administered in the first treatment
step may be administered by any conventional technique used in the
art (e.g., oral, subcutaneously, intralymphatically,
intraperitoneally, intravenously, or intramuscularly). In one
embodiment of the invention, the cytotoxic chemical compound
(preferably BCNU, cisplatin, or cyclophosphamide) is administered
intravenously. Likewise, squalamine can be administered by any
conventional administration method known in the art, such as those
mentioned above. Subcutaneous injections of squalamine one or two
times a day are used in one embodiment of this invention.
Intravenous administration of squalamine one or two times a day are
used in another embodiment of the present invention.
The first treatment procedure with the cytotoxic chemical compound
may take place prior to the second treatment procedure (using
squalamine), after the second treatment procedure, or at the same
time as the second treatment procedure. Furthermore, the first
treatment procedure may be completed before the second treatment
procedure is initiated (or vice versa). In one embodiment of the
invention, the first treatment procedure is a one time intravenous
administration of a cytotoxic chemical compound (e.g., BCNU,
cisplatin, or cyclophosphamide), and the second treatment procedure
involves daily subcutaneous injections of squalamine.
In a second method for treating a tumor according to the invention,
the first treatment procedure is a radiation treatment, which may
be one or more conventional radiation modalities, using a
conventional radiation treatment regimen known to those skilled in
the art. The tumor is exposed to radiation in this first treatment
procedure. In a second treatment procedure, an effective amount of
squalamine is administered to treat the tumor. Appropriate timing
of the radiation treatment procedure with respect to the squalamine
treatment regimen can be determined by those skilled in the art
through routine experimentation in order to provide effective tumor
treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other advantageous features of the invention will be more
fully appreciated when considered based on the following detailed
description and the attached drawings, wherein:
FIG. 1 shows the general structural formula of squalamine;
FIG. 2 shows a general overview of the angiogenesis process;
FIG. 3 is a drawing used to illustrate the sodium hydrogen
exchanger (NHE) process;
FIG. 4 illustrates the effects of conventional amilorides on
inhibiting various isoforms of mammalian NHEs;
FIGS. 5a and 5b illustrate the effect of squalamine on NHE isoform
3 (NHE3) and NHE1 inhibition, respectively;
FIGS. 6a to 6c show the results of a pharmacokinetic study relating
to squalamine;
FIG. 7 illustrates squalamine distribution in various tissues after
i.v. administration;
FIG. 8 shows an angiogenesis index using squalamine as determined
in the rabbit corneal micropocket assay;
FIG. 9 shows the inhibitory effect of squalamine on growth of
endothelial cells as compared to tumor cell lines;
FIG. 10 illustrates survival test results using squalamine in a
glioma lethality study with a rat 9L glioma introduced into the
brains of healthy rats;
FIG. 11 shows the survival of mice carrying human MX-1 breast tumor
xenografts and treated with squalamine subsequent to
cyclophosphamide treatment;
FIG. 12 depicts the inhibition of a human lung adenocarcinoma
(H460) in a mouse xenograft-combination therapy study with
squalamine and cisplatin; and
FIG. 13 illustrates the number of lung metastases following various
chemotherapeutic treatment procedures in mice with subcutaneous
implanted Lewis lung carcinomas.
DETAILED DESCRIPTION OF THE INVENTION
Squalamine has been recognized to have angiogenesis inhibiting
activity, i.e., it inhibits the formation of blood vessels.
Therefore, it is believed that squalamine, as an antiangiogenic
agent, will be effective in treating certain diseases or ailments
which depend on neovascularization. For example, squalamine may be
used for treating such disparate conditions as solid tumor cancers,
macular degeneration, diabetic retinopathy, psoriasis, or
rheumatoid arthritis, all of which require a separate and new blood
flow.
In addition, squalamine can selectively inhibit certain
sodium/proton exchangers (also called "NHEs" or "proton pumps" in
this application). Several different isoforms of NHE are known to
exist in mammals (e.g., NHE1, NHE2, NHE3, NHE4, and NHE5).
Squalamine has been found to specifically inhibit NHE3 and not NHE1
or NHE2. Accordingly, squalamine may be used for treating
proliferation or activation dependent conditions which rely on the
function of NHE3, such as cancer, viral diseases, and ischemic
reprofusion injury.
Further studies with squalamine and NHE have demonstrated that
squalamine acts on a very specific portion of the NHE3, namely the
76 carboxyl-terminal amino acids of the molecule. If this portion
of the NHE3 molecule is removed, squalamine has virtually no effect
on the activity of the molecule, even though the molecule is still
active as a sodium/proton exchanger.
Applicants have discovered still further uses of squalamine.
Specifically, applicants have found that squalamine in combination
with conventional cancer treating agents, e.g., cytotoxic chemical
compounds and radiation treatments, will decrease the size and
growth of tumors. Even more significantly, applicants have found
that the combination decreases the growth rate of highly
proliferative CNS tumors, lung tumors, and breast tumors and can
confer survival advantages.
In the practice of this aspect of the invention, a cytotoxic
chemical compound is used in a first tumor treatment procedure, and
squalamine is used in a second tumor treatment procedure. The first
and second treatments may be administered in any time sequence or
even simultaneously. In another embodiment, two or more cytotoxic
chemical agents may be administered simultaneously or sequentially
in the first treatment process.
The cytotoxic chemical compound(s) used in the first treatment
procedure may be any conventional agent, but it is preferably one
of the following agents: a nitrosourea, cyclophosphamide,
adriamycin, 5-fluorouracil, paclitaxel and its derivatives, and
cisplatin and related platinum compounds. These materials are
conventional cancer treating agents which are known to those
skilled in this art, as set forth in Medical Oncology, supra. One
particularly preferred nitrosourea is BCNU, which is also known as
"carmustine" or "1,3-Bis(2-chloroethyl)-1-nitrosourea."
Cyclophosphamide also is known as
N,N-Bis-(2-chloroethyl)-N'-(3-hydroxypropyl)phosphordiamidic acid
cyclic ester monohydrate. Adriamycin also is known as
doxorubicin.
Paclitaxel is available under the tradename "Taxol." Various
derivatives of paclitaxel may be used in accordance with the
invention, such as taxotere or other related taxanes. Cisplatin,
another of the cytotoxic chemical compounds which may be used in
accordance with the invention, also is known as
cis-Diamminedichloroplatinum. Those of ordinary skill in the art
would be familiar with other specific cytotoxic agents that could
be used in the process of the invention.
There are no limitations on the chemotherapeutic agent that can be
used in this invention. Other conventional chemotherapeutic agents
that can be used with squalamine in the process of the invention
include methotrexate, thiotepa, mitoxantrone, vincristine,
vinblastine, etoposide, ifosfamide, bleomycin, procarbazine,
chlorambucil, fludarabine, mitomycin C, vinorelbine, and
gemcitabine.
The first and/or second treatments may be administered by any
suitable technique, such as oral, "s.q.," "i.p.," "i.m.," "i.l.,"
or i.v." In this application, the terms "s.q.," "i.p.," "i.m.,"
"i.l.," and "i.v." will be used to refer to subcutaneous
administration of squalamine or other substances, intraperitoneal
administration of squalamine or other substances, intramuscular
administration of squalamine or other substances, intralymphatic
administration of squalamine or other substances, and intravenous
administration of squalamine or other substances, respectively.
In one embodiment, BCNU is delivered to a patient first as a one
time intravenous dosage, and thereafter squalamine is injected s.q.
twice daily. In another embodiment, cyclophosphamide is the
cytotoxic agent. In another embodiment, cisplatin is the cytotoxic
agent. If appropriate, the cytotoxic chemical compound and the
squalamine may be delivered simultaneously by a common
pharmaceutical carrier (e.g., one injection including both
squalamine and the cytotoxic chemical compound). Other appropriate
combinations of administration techniques may be used without
departing from the invention. Those skilled in the art will be able
to ascertain the appropriate treatment regimens, depending on the
cytotoxic chemicals used, the dosages, etc., through routine
experimentation.
The squalamine treatment procedure in accordance with the invention
also may be used with radiation treatment (e.g., cobalt or X-ray
treatment) as the first treatment procedure. In this embodiment of
the invention, the first treatment procedure is a radiation
treatment, and the second treatment procedure is squalamine
administration. Radiation treatments can proceed on a schedule in
combination with the squalamine treatments to provide optimum
results. Such scheduling of the treatment procedures can be
ascertained by the skilled artisan through routine experimentation.
Any conventional radiation treatment, such as those described in
Medical Oncology, supra., may be used without departing from the
invention. In addition to radiation and squalamine treatments, the
tumor also may be treated with one or more cytotoxic chemical
compounds in a third treatment procedure.
The invention will be described below in terms of various specific
examples and preferred embodiments. These examples and embodiments
should be considered to be illustrative of the invention, and not
as limiting the same.
I. PHYSIOLOGICAL PROPERTIES OF SQUALAMINE
A. Antiangiogenic Activity
Squalamine has been demonstrated to be useful as an antiangiogenic
agent, i.e., squalamine inhibits angiogenesis. Angiogenesis, the
process of forming new blood vessels, occurs in many basic
physiological processes, such as embryogenesis, ovulation, and
wound healing. Angiogenesis also is essential for the progression
of many pathological processes, such as diabetic retinopathy,
inflammation, and malignancy (tumor development). In view of its
antiangiogenic properties, squalamine may be used for treating
various ailments and conditions which depend on angiogenesis, such
as those identified above.
Angiogenesis is a multiple step process which is schematically
illustrated in FIG. 2. First, endothelial cells must become
activated, for example, by attaching a growth factor such as
vascular endothelial growth factor ("VEGF") or basic-fibroblast
growth factor ("b-FGF"). The cells then move, divide, and digest
their way into adjacent tissue through the extracellular matrix.
The cells then come together to form capillaries and lay down new
basement membrane. This angiogenesis process is illustrated in the
upper portion of FIG. 2. Each of these development stages during
angiogenesis is important and may be affected by antiangiogenic
agents.
Certain compounds which are believed to be antiangiogenic compounds
(e.g., matrix metalloproteinase inhibitors, such as minocycline,
SU101 or marimistat) act at later stages in this multistep
angiogenesis process. These compounds will be referred to as
"downstream" angiogenesis inhibitors. For a discussion of matrix
metalloproteinase inhibitors, please refer to Teicher, Critical
Reviews in Oncology/Hematology, Vol. 20 (1995), pp. 9-39. This
document is entirely incorporated herein by reference. In contrast
to these known antiangiogenic compounds, squalamine acts at a very
early stage in the process by inhibiting the cell activation action
of growth factors, i.e., it is an "upstream" angiogenesis
inhibitor. As shown in FIG. 2 (toward the bottom), squalamine
inhibits the sodium-proton pumps that are normally active and
activated by the growth factors. Inhibition of the proton pump
places the cell in a quiescent state, and, in this way, capillary
formation and angiogenesis is impeded. In effect, the growth factor
signal is aborted in the presence of squalamine.
B. Capillary Regression Activity
In addition to antiangiogenic characteristics, squalamine has been
shown to have a capillary regression effect in newly formed
capillaries. A one time dose (100 ng) of squalamine was applied to
capillary beds of young chick embryos that were 2-3 days old. After
five minutes, this dose of squalamine appeared to have little
effect on the capillary beds. In twenty minutes, however, the
capillary bed appeared to be disappearing (i.e., the vessels
appeared to be closed off). After forty minutes, additional
capillary regression was observed.
The capillary bed also was observed after sixty minutes. At this
time, it was noted that some of the capillary vessels were
beginning to re-appear, but only the more major vessels were
re-appearing. The small vessels were not re-appearing at that time.
Four to five days after the one time squalamine treatment, the
effect of the squalamine dose was no longer apparent, but newly
formed capillaries in the embryos remained susceptible to
squalamine induced regression for a limited time while they were
newly formed.
From this test, applicants concluded that squalamine-induced
capillary regression is reversible, at least with respect to
certain capillaries. It also was concluded that squalamine is more
effective against small microcapillary blood vessels (i.e., the
microvascular bed) as compared to the major blood vessels. Close
histological examination of chick microvessels exposed to
squalamine revealed vessel occlusion was due to shrinkage of
endothelial cell volumes in cells wrapped around the vessel lumen.
The applicants postulate that occlusion or regression of small
blood vessels by squalamine significantly contributes to the
ability of squalamine to impede the flow of nutrients and growth
factors into tumors and thereby slows or blocks the rate of growth
of the tumors.
C. NHE Inhibitory Activity Of Squalamine
Cell growth and division is necessary for blood vessel and
capillary growth and formation. Capillary formation requires a
specific extracellular matrix. The NHE antiporter system of a cell
is connected to the extracellular matrix. Activation of the NHE
antiporter is necessary to induce cell growth, and interference
with the NHE antiporter interrupts the matrix signal and interferes
with cell growth. When endothelial cell growth is interrupted,
capillary growth is impeded.
The NHE antiporter of cells may be activated in different ways. For
example, insoluble fibronectin activates the NHE antiporter by
clustering and immobilizing Integrin .alpha..sub.v .beta..sub.1,
independent of the cell shape (the growth of anchorage-dependent
cells requires both soluble mitogens and insoluble matrix
molecules). In addition, the attachment of stimuli to the
extracellular matrix or cell attachment events involving viruses
also activate the NHE antiporter.
When activated, the NHE antiporter induces cell growth by
regulating the pH of the cell. As shown in FIG. 3, the
chloride-bicarbonate exchanger and NHE are complementary pH
regulators in cells. The chloride-bicarbonate exchanger makes the
cell become more alkaline, while NHE contributes to the control of
hydrogen ion concentration in the cell. When the NHE is inhibited,
the cells become acidic (lower pH) and growth stops. This does not
mean that the cell dies; it means only that the cell enters a
quiescent state (i.e., it does not divide). If the cell returns to
a normal pH, growth may resume. When the NHE is activated, the cell
becomes more alkaline (higher pH), it pumps out protons, and growth
proceeds. Interaction of various modulatory factors (e.g., serum
components, secondary messengers, etc.) with one portion of the
cytoplasmic region of NHE activates the antiporter, while
interaction with another portion inhibits the antiporter. These
portions of NHE are described in Tse, et al., "The Mammalian
Na.sup.+ /H.sup.+ Exchanger Gene Family--Initial Structure/Function
Studies," J. Am. Soc. Nephr., Vol. 4 (1993), pg. 969, et seq. This
article is entirely incorporated herein by reference.
Sodium-proton pumps (NHEs) are responsive to different growth
stimuli which activate the pump. As noted above in connection with
FIG. 2, the proton pump may be activated by attachment of growth
factors (e.g., VEGF and b-FGF) to the cell. Additionally, as shown
in FIG. 3, other stimuli, such as virus attachment, addition of
various mitogens, sperm attachment to an egg, etc, also can cause
NHE activation and alkalinization of the cell. Attachment of these
stimuli to the extracellular matrix activates the NHE antiporter of
the cell and induces cell growth.
At least five different mammalian isoforms of NHE exist, and each
has a distinct tissue distribution. Nonetheless, all act in the
same manner. NHE1 is the antiporter found in all tissues. NHE2 and
NHE3 are more restrictive in their tissue distribution.
The effect of squalamine on NHE activity was measured to determine
which isoforms of NHE were affected by squalamine. NHE activity can
be measured under various different cellular conditions. Acid
loading a cell activates all of the antiporters and permits
measurement of NHE. NHE activity also can be measured after growth
factor stimulation of the cell. Additionally, the NHE activity can
be measured when the cell is in an unstimulated state, because the
antiporters, even if unstimulated, continue to function at a slow,
but non-zero rate. In each of these cellular conditions, NHE
activity usually is measured in the absence of bicarbonate.
Amilorides, which are the classic inhibitors of activated NHE
antiporters and which act as direct competitive inhibitors of
Na.sup.+ ion binding to NHE, do not turn off the antiporter
activity in unstimulated cells. As illustrated in FIG. 4, amiloride
and amiloride analogues specifically act against NHE1 over NHE2 or
NHE3. NHE3 in particular is relatively resistant to inhibition by
the amilorides. In contrast to the amilorides, when NHE1 activity
was measured in unstimulated melanoma cells, applicants found that
squalamine substantially down regulates the activity of the
antiporter.
The following describes the test used to determine that squalamine
inhibits NHE3, but not NHE1 or NHE2. NHE deficient fibroblast cells
(PS120) transfected with an individual human NHE gene were loaded
with a pH sensitive dye
2'7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). NHE
activity was measured by spectrofluorometric methods using this dye
and by amiloride sensitive isotopic .sup.22 Na.sup.+ cellular
uptake. The cells were acidified by exposure to ammonium chloride
in the absence of sodium to eliminate sodium and deactivate the
proton pumps. The ammonium chloride was washed out by exposing the
cells to tetramethyl ammonium chloride in bicarbonate free medium.
The cells were consequently acidified, but in the absence of
sodium, the NHE ion pumps did not activate. For this test, as shown
in FIGS. 5a and 5b, 7 .mu.g/ml of squalamine was added to the cells
in each case. Sodium then was added back at various concentrations
(see the abscissa of FIGS. 5a and 5b) to drive the antiporters
(human NHE3 in FIG. 5a and human NHE1 in FIG. 5b). The antiporters
were driven at different rates, as evidenced by the cellular pH
change rate, depending on the amount of sodium added. As shown in
FIG. 5a, when measuring the effect of squalamine on the human NHE3
antiporter, the pH change rate was lower in the squalamine treated
cells than the pH change rate in the control group (without
squalamine). This indicates that squalamine inhibits human NHE3. In
FIG. 5b, however, there is no effective difference in the pH change
rate between the squalamine treated samples and the control when
measuring the human NHE1 antiporter. From these tests, applicants
concluded that squalamine inhibits human NHE3, but not human NHE1.
Additionally, in similar tests, it was found that rabbit NHE1 and
NHE2 are not affected by squalamine, but rabbit NHE3 is inhibited
by squalamine treatment.
In the transfected cells used in this test, it took at least 30
minutes before the NHE3 inhibition effect induced by squalamine was
observed. Thus, squalamine did not act like the classic NHE
inhibitor amiloride or analogues of amiloride, which are direct
competitive inhibitors for sodium and, therefore, act rapidly as
NHE inhibitors. Furthermore, it was observed that the NHE
inhibiting effect of squalamine occurred in the absence of lactase
dehydrogenase (LDH) leakage from the cell. Because LDH leakage is a
non-specific marker of cytotoxicity, it was concluded that
squalamine does not have a general cytotoxic effect.
This NHE3 inhibiting activity of squalamine has been mapped to the
76 C-terminal amino acids on the NHE3 molecule. If the 76
C-terminal amino acids of rabbit NHE3 are removed from the
molecule, squalamine has been found to have virtually no effect on
the activity of the molecule, while the molecule remains active as
a sodium/hydrogen exchanger. Thus, the 76 C-terminal amino acids of
NHE3 are the site of inhibition by squalamine. It is believed that
the squalamine effect on these accessory proteins of NHE3 is tied
to an inhibitory effect on tyrosine kinase-dependent activity,
although applicants do not wish to be bound by any specific theory
of operation.
As noted above, it has been concluded that squalamine inhibits NHE3
and not NHE1. This inhibitory effect of squalamine, however, has
been found to work in a manner different from classical and known
NHE3 inhibitors. In contrast to squalamine, other inhibitors of
NHE3 (e.g., amiloride, amiloride analogues, genestein, calmodulin,
and protein kinase C) also inhibit NHE1. Such inhibitors affect
only the absolute number of protons that can be secreted by the
cell (i.e., "V.sub.max "), if one looks at the kinetic
characteristics of the inhibition. Squalamine, on the other hand,
not only inhibits V.sub.max, but it also forces the cell to fall to
a lower pH, as evidenced by a reduction in the Km value. Note the
following Table 1, which correlates to data collected in the test
of FIG. 5a.
TABLE 1 ______________________________________ Squalamine (7
.mu.g/ml) Control ______________________________________ Km 0.338
0.595 n 1.88 1.22 V.sub.max 1282 2958
______________________________________
Thus, squalamine inhibits NHE with nonallosteric kinetics (i.e.,
nonclassical allosteric inhibition). In additional tests, it also
was found that squalamine (at a 1 hour pretreatment) decreased the
V.sub.max of rabbit NHE3 in a concentration dependent manner (13%,
47%, and 57% with 1, 5, and 7 .mu.g squalamine/ml, respectively).
This observed squalamine effect on the V.sub.max was time
dependent, with a maximum effect occurring at one hour exposure.
The observed effect was fully reversible within three hours after
removing the cells from the medium.
In view of the test results relating to the effect of squalamine on
NHE3, applicants believe that NHE3 is important in maintaining
homeostasis of the unstimulated cell. The applicants further
believe that prevention of cellular activation by squalamine,
especially activation of endothelial cells or precursor cells which
participate in formation of new blood vessels during
pathophysiological vascularization (such as during tumor growth),
is the mechanism through which squalamine inhibits tumor
growth.
Applicants have further observed that squalamine changes
endothelial cell shape. This suggests that transport proteins which
control cell volume and shape may be a squalamine target.
Additional testing of squalamine has indicated that squalamine
inhibited brush border membrane vesicle (BBMV) NHE only when the
tissue was pretreated with squalamine (51% inhibition at 30 minutes
exposure). Direct addition of squalamine to PS120 fibroblasts
during measurement of the exchanger activity had no effect.
D. Pharmacokinetic Studies Of Squalamine
A pharmacokinetic study of squalamine was performed to ascertain
the residence time of squalamine in the body. FIGS. 6a to 6c
illustrate the test results where squalamine was administered
subcutaneously (50 mg/kg, FIG. 6a), intraperitoneally (dose 240
.mu.g; 10 mg/kg, FIG. 6b), and intravenously (10 mg/kg, FIG. 6c).
The half-life of squalamine when given intravenously (FIG. 6c) was
acceptable (35 minutes), but it was even higher when it was
administered intraperitoneally (FIG. 6b, half-life=172 minutes) and
subcutaneously (FIG. 6a, half-life=5.6 hours).
In addition to these squalamine half-life tests, applicants have
tested to ascertain the distribution of squalamine in a mouse after
intravenous administration. FIG. 7 illustrates the distribution of
squalamine in mouse tissue two hours after i.v. administration.
Some squalamine is contained in most of the tissues, with most of
the squalamine concentrating in the liver and the small intestine.
The test results shown in FIG. 7 indicate good squalamine
distribution. Notably, however, not much squalamine is present in
brain tissue. From this, applicants conclude that squalamine
probably does not cross the brain/blood barrier. In treating brain
tumors, it is believed that the squalamine acts on the endothelial
cells in the brain, and in this way, it need not cross the
brain/blood barrier
The following examples describe more detailed experiments used to
test the antiangiogenic characteristics of squalamine in the
process of the invention.
EXAMPLE 1
Rabbit Corneal Micropocket Assay
In determining whether a compound is antiangiogenic, the rabbit
corneal micropocket assay is an accepted standard test. In this
test, an incision is made in one rabbit cornea, and a stimulus is
placed in the incision. The stimulus is used to induce blood vessel
formation in the normally avascular corneal region. As one example,
a solid tumor in a polymeric matrix can be placed in the cornea as
the stimulus because the tumor will release a number of angiogenic
growth factors to stimulate new capillary growth. The tumor-derived
angiogenic growth factors stimulate the endothelial cells at the
scleral junction in the eye to initiate blood vessel growth toward
the stimulus. A second polymer pellet (e.g., an ethylene/vinyl
acetate copolymer) is placed between the scleral junction and the
stimulus. This polymer pellet is either empty (a negative control
test pellet), or it contains a compound whose antiangiogenic
characteristics are to be tested. The polymer pellet is used to
provide a controlled release of the material to be studied. Because
of the avascular cornea background in the rabbit cornea, one can
visually assess the results qualitatively. In addition, the number
of blood vessels can be counted and their length, etc., can be
measured to provide a more quantitative evaluation of the
results.
The VX2 rabbit carcinoma was implanted in 26 rabbit eyes, in the
normally avascular corneal region, to act as an angiogenesis
stimulus. Squalamine was incorporated into a controlled release
ethylene/vinyl acetate copolymer (20% squalamine and 80% polymer by
weight). The loaded polymer pellets were placed in 13 of the
corneas to provide a sustained local release of squalamine. Polymer
blanks were provided in the remaining 13 eyes as a control. In this
manner, one eye of each rabbit served as the squalamine test eye
and the other eye of the same rabbit served as the control eye. The
eyes were examined weekly using a slit lamp stereomicroscope for
three weeks after tumor implantment, and the Angiogenesis Index
("AI") was calculated (this calculation will be described in more
detail below with reference to FIG. 8). The squalamine loaded
polymer was found in vitro to release active squalamine throughout
the treatment period. After the test, the corneas were examined
histologically.
Using this test, squalamine was found to be a potent inhibitor of
tumor induced capillary formation. Fewer blood vessels were
observed in the cornea treated with squalamine as compared to the
control cornea, and these vessels were generally shorter than the
vessels in the control cornea.
Some of the corneas were then sectioned to observe the effect of
squalamine on the tumor cells themselves. The untreated control
corneas had many vessels in and adjacent to the tumor. The tumors
in the squalamine-treated corneas were still viable (i.e., the
tumors were not dead), but there was essentially no vasculature
associated with those tumors. Thus, the squalamine-treated tumors
had greatly diminished vascularity as compared to the corresponding
control tumor sections. These findings suggested that squalamine
works against the blood vessels, and not against the tumor
itself.
FIG. 8 shows a graphical representation of the results of the
rabbit cornea micropocket assay test. To provide a quantitative
evaluation, the Angiogenesis Index ("AI") of each eye was
determined. To determine the Angiogenesis Index, first the vessel
density ("D.sub.vessel ") in an eye was graded on a 0-3 scale as
follows:
TABLE 2 ______________________________________ D.sub.vessel Value
Determinations D.sub.vessel Value Visual Observation
______________________________________ 0 No vessels present 1 1-10
vessels present 2 >10 vessels present, but loosely packed 3
>10 vessels present, packed densely
______________________________________
The vessel length ("L.sub.vessel ") was then measured in each
cornea. The vessel length is the length of the longest vessel
measured from the cornea-scleral junction to the distal edge of the
longest vessel growth. The Angiogenesis Index then is determined
from these measurements by the following equation:
FIG. 8 shows the mean Angiogenesis Index for each group of corneas
(squalamine treated and untreated) in the rabbit cornea micropocket
assay after 1, 2, and 3 weeks. As shown in the figure, squalamine
was very inhibitory to the growth of new blood vessels. The
squalamine treated eyes showed a significantly reduced AI value as
compared to the untreated eyes (37% reduced at Day 14 (p=0.05,
Wilcoxon rank sum test) and 43% reduced at Day 21 (p<0.01). This
data illustrates that squalamine inhibits tumor induced growth of
new blood vessels or capillaries over a long time period. More
specifically, squalamine exhibits high antiangiogenic activity even
after three weeks.
EXAMPLE 2
Squalamine Does Not Cause Inflammation
In the rabbit corneal micropocket assay test, if the rabbit cornea
becomes inflamed, this inflammation can lead to the formation of
new blood vessels in the cornea. Such inflammation would skew the
test results. Therefore, tests were conducted to determine whether
squalamine, in and of itself, was responsible for any inflammatory
response in the cornea. Several non-bioresorbable ethylene/vinyl
acetate copolymer pellets were loaded with different concentrations
of squalamine, namely, 2%, 10%, and 20% squalamine, by weight.
These pellets were then placed in rabbit corneas which did not
include an angiogenic stimulus. Squalamine did not induce
inflammation at any of these concentrations. Thus, squalamine does
not lead to the generation of new blood vessels by inflaming the
cornea.
EXAMPLE 3
Squalamine Use in Brain Tumor Treatment
The rabbit corneal micropocket assay test results suggested to
applicants that squalamine may be a potent antiangiogenic agent
that inhibits neovascularization. Recognizing that the exponential
growth of solid tumors in the brain is dependent on
neovascularization, applicants assessed the activity of squalamine
in an animal model on the growth of solid tumors in the brain.
Of solid brain tumors, malignant gliomas are the most common form
of cancerous tumors. These tumors are the third leading cause of
death from cancer in young adults between the ages of 15 and 34.
Malignant gliomas are characterized by their ability to induce the
normally quiescent brain and/or CNS endothelial cells into a highly
proliferative and invasive state. The gliomas express vascular
endothelial growth factor ("VEGF") and other growth factors which
stimulate inducible receptors on CNS endothelial cells in a
paracrine manner (i.e., the VEGF originates from the tumor cell and
stimulates the endothelial cells). The CNS endothelial cells
subsequently initiate angiogenic invasion and thus provide
nourishment of the glioma. Applicants tested the antiangiogenic
activity of squalamine against gliomas by testing (1) its ability
to selectively inhibit VEGF-mediated stimulation of endothelial
cells and (2) its effect against experimental murine glial
tumors.
In vitro tests were first performed to determine that squalamine
acts specifically on endothelial cells. Applicants used endothelial
cells because such cells are involved in the early steps of
angiogenesis, as described above in conjunction with FIG. 2.
Specifically, tumor angiogenesis is a series of sequential and
overlapping steps. First, the endothelial cells activate and
proliferate. Then, proteolytic enzymes are produced and the cells
migrate. New basement membranes must then be generated. In this
manner, new blood vessels are generated and tumor size
increases.
In conducting this in vitro analysis, the following cell lines were
tested: (a) bovine retinal endothelial cells; (b) 9L and C6 rat
glioma cells; (c) human H80 glioma cells; and (d) VX2 rabbit
carcinoma cells (the same type as the tumors implanted in the
rabbit corneal micropocket assay test described above). The
endothelial mitogen which was used in this analysis was VEGF at a
concentration of 20 ng/ml.
The cells were allowed to attach overnight to tissue culture plates
containing an optimized growth media. Following attachment, the
cells were exposed to solvent only or to increasing concentrations
of squalamine (0, 10, 20, 30, 60, and 90 .mu.g squalamine/ml). Cell
growth was counted daily for three days using a Coulter Counter. A
total of 10,000 cells per well were plated and each experimental
concentration was tested in quadruplicate. The results were then
averaged. The bovine retinal endothelial cells were grown and
treated in an identical manner to the other cell lines, except that
the growth of these cells was measured after the addition of 20
ng/ml of human recombinant VEGF to the cells prior to the
squalamine treatment.
Cell proliferation by all tumor lines and by endothelial cells not
treated with VEGF was statistically unaffected after exposure for
24 and 48 hours to squalamine concentrations up to 30 .mu.g/ml.
Growth of the VEGF-stimulated endothelial cells, however, was
significantly reduced by squalamine at these same times in a
concentration dependent manner. Percentage endothelial cell growth
inhibition (%I) was determined by the following equation: ##EQU1##
The following Table shows the results at 48 hours for the
VEGF-stimulated endothelial cell line.
TABLE 3 ______________________________________ Percent Inhibition
Data Squalamine Conc. % Inhibition (average)
______________________________________ 10 .mu.g/ml 38% (p <
0.01) 20 .mu.g/ml 57% (p < 0.001) 30 .mu.g/ml 83% (p < 0.001)
______________________________________
Additional data is illustrated in FIG. 9. This figure shows the
growth of the various cell lines as a percentage of the growth in
the control groups for in vitro administration of squalamine at 30
.mu.g/ml after 1, 2, and 3 days. As shown in FIG. 9, growth is
reduced for the VEGF-stimulated endothelial cells specifically,
while the growth in the other cell lines (H80, C6, and VX2) is not
dramatically affected.
Based on this information, applicants concluded that squalamine
dramatically and specifically inhibits VEGF-stimulated growth of
endothelial cells in vitro. Thus, squalamine is a potent inhibitor
of tumor-induced angiogenesis, and this effect appears to be
precipitated through specific inhibition of endothelial cell
proliferation induced by VEGF. Thus, squalamine is believed to be
well suited for reducing or diminishing the neovasculature induced
by tumors for use in tumor specific antiangiogenic therapy.
In addition to inhibiting VEGF-stimulated growth of endothelial
cells, squalamine also has been found to interfere with growth
stimulation in human brain capillary endothelial cells induced by
b-FGF, PDGF.sub.bb, scatter factor (HGF or hepatocyte growth
factor), conditioned tumor media, and human brain cyst fluid. Thus,
as the tumor puts out a variety of different growth factors,
squalamine has an inhibitory effect on several.
In view of these test results, applicants tested squalamine in an
animal model for brain cancer. To test the effect of squalamine on
tumors located in the brain, small sections (1 mm.sup.3) of
existing rat gliomas were taken from rat flanks where they were
being maintained and were implanted into the rat brains in two
groups of rats. Thus, in this model, the tumors were viable when
placed in the rat brain. Three days after implantation, and after
some vasculature had developed, treatment with 20 mg/kg/day of
squalamine (i.p.) was initiated in one group of rats. The control
animals ("vehicle control" in FIG. 10) were given the carrier
vehicle only (no squalamine), and the other animals were treated
with squalamine ("Squalamine" in FIG. 10). As shown in the figure,
the animals treated with squalamine had a 38% increase in mean
survival time (x=24.9 days v. x=18.0 days). FIG. 10 further
illustrates that in this animal model, the squalamine treated rats,
in general, had an increased survival time.
A squalamine toxicity test was performed in another animal model.
Conventional cytotoxic chemical compounds are quite toxic. For
example, BCNU, which is a conventional chemotherapy agent, has a
cumulative toxicity effect. For this reason, it is administered
only one time to a patient. The use of BCNU is described on pages
304 and 305 of Calabresi in Medical Oncology, supra. In order to
test the toxicity of squalamine, a group of rats was given a daily
squalamine dose of 20 mg/kg/day (i.p.) for more than 30 days and
maintained for up to 200 days following dosing. The animals in this
study remained healthy. This result indicates that squalamine has
little or no toxicity.
EXAMPLE 4
Squalamine Use With Conventional Cancer Treatments
As described above, squalamine is an upstream inhibitor of the
angiogenesis process by inhibiting the activation of endothelial
cells after growth factor interaction. Because of its angiogenesis
inhibiting properties, squalamine has been demonstrated to be
effective in treating solid tumors which rely on neovascularization
to proliferate. Applicants tested to determine whether beneficial
results could be obtained when treating tumors by combining a
squalamine treatment (an upstream angiogenesis inhibitor) with a
conventional cancer treatment using an alkylating agent.
a. The Squalamine 9L Glioma Flank Study
Four groups of rats (twenty total Fisher 344 rats, 200 g) were
given s.q. transplants of 1 mm.sup.3 9L gliosarcoma tumors (9L
glioma) on Day 0. The tumors were implanted in the rat flanks to
avoid complications relating to adequate brain levels of
squalamine. Randomization and treatment began on Day 5 according to
the following scheme:
TABLE 4 ______________________________________ Treatment Conditions
Group No. Treatment ______________________________________ 1 Saline
(control group) 2 One time dose of 14 mg/kg BCNU given i.p. on Day
5 3 Squalamine - 20 mg/kg given s.q. B.I.D..sup.1 4 One time dose
of 14 mg/kg BCNU given i.p. on Day 5 and daily injection of
squalamine - 20 mg/kg given s.q. B.I.D- beginning on Day 5.
______________________________________ .sup.1 The term "B.I.D"
means that the component is administered twice a day (10 mg/kg
given at two different times each day).
On Day 25 or 26 after tumor implantation, the tumor size was
measured directly. The tumor size (i.e., its volume "V") was
estimated based on volumetric calculations determined from the
measured length ("L"), width ("W"), and height ("H") of the tumor
(V.sub.tumor spheroid .apprxeq.0.5.times.L.times.W.times.H). Table
5 summarizes the results. The tumor volumes shown in Table 5
represent the mean tumor volumes for each treatment group for those
animals that survived to the end of the experiment.
TABLE 5 ______________________________________ Tumor Values Mean
Tumor volume % Reduction (based Group No. No. of Animals (mm.sup.3)
on control volume) ______________________________________ 1 5
18,324 -- 2 6 2,547 86.1% 3 5 3,347 81.7% 4 4 38 99.8%
______________________________________
Table 5 illustrates the advantageous results achieved when treating
tumors with the combination of squalamine and the nitrosourea BCNU
(Group 4). A 99.8% reduced mean tumor size was observed when
treating with both squalamine and BCNU in this group. Table 5
further shows that squalamine alone (Group 3) was effective in
treating the tumor. The tumor size was reduced by 81.7% in Group 3,
as compared to the control group.
Applicants conclude that the use of squalamine in combination with
conventional cytotoxic chemical compounds can slow or halt the
spread of brain cancers. The tumor itself shrinks and becomes
necrotic. It is expected that combined squalamine and cytotoxic
chemical treatment will extend survival. Thus, this treatment
potentially will allow management of brain cancers.
b. Squalamine Use in Breast Tumor Treatment
The human MX-1 breast cancer line has previously been used to
document in vivo activity of cyclophosphamide and other cytotoxic
chemotherapeutic compounds either as single agents or in
combination (T. Kubota, et al., Gann 74, 437-444 (1983); E.
Kobayashi, et al., Cancer Research 54,, 2404-2410 (1994); M.-C.
Bissery, et al., Seminars in Oncology 22 (No. 6, Suppl. 13), 3-16
(1995)). These documents each are entirely incorporated herein by
reference. Squalamine was examined as adjunctive therapy following
a single 200 mg/kg dose of cyclophosphamide. The cyclophosphamide
was injected on day 14 following implantation of the tumor, at a
time when the tumors measured 65-125 .mu.l. The cyclophosphamide
caused partial regression in all animals and complete regression in
a small fraction of the animals. The animals were then randomized
to three treatment arms (each n=27): vehicle dosing only
(Intralipid); squalamine given 10 mg/kg/day in Intralipid; and
squalamine given 20 mg/kg/day in Intralipid for five days a week.
Animals whose tumors exceeded 2 grams at any time during the
experiment were euthanized. The experiment was continued for 90
days after initiating squalamine treatment to ensure that only mice
experiencing long-term cures were still alive. The high dose
squalamine was discontinued after five weeks of treatment because
of animal weight loss and potential toxicity concerns, so these
animals did not receive squalamine for the last eight weeks of the
experiment. The low dose squalamine treatment produced a
significant (P<0.01) inhibition in the rate of progression of
the breast tumors at all times examined (FIG. 11). The high dose
squalamine treatment produced significant (P<0.05) delay in
progression of the breast tumors only at 30 days post-initiation
(i.e., only while squalamine was still being given), but high dose
squalamine also doubled the long-term cure rate in these animals
compared to controls which received cyclophosphamide alone (FIG.
11). Examination of the history of the long-term cure animals which
received cyclophosphamide and high dose squalamine revealed that
the additive effects of squalamine were manifested within two weeks
after starting squalamine treatment.
c. Squalamine Use in Lung Tumor Treatment
Studies in a nude mouse xenograft model of lung cancer have been
carried out using several human lung cancer lines which differ in
their growth rate. The data collected show that squalamine has
synergistic activity in combination with cisplatin (e.g., FIG. 12).
The experimental lung cancer model design involves subcutaneous
injection of 5.times.10.sup.6 tumor cells followed by a single
injection of the chemotherapeutic drug on day 3 or 4. Daily
intraperitoneal squalamine injections with 20% Intralipid as a
vehicle began the following day for some groups of mice and
continued until the experiment was terminated 7-14 days later.
Groups of mice receiving squalamine alone started receiving the
aminosterol on the same day as aminosterol treatment in the
combination chemotherapy groups. Tumor volumes were then determined
at termination of the experiment and compared. It was found for
both the aggressively growing H460 human lung adenocarcinoma line
and for the more slowly growing Calu-6 human lung adenocarcinoma
line that squalamine had minimal effects on tumor growth as a
monotherapeutic agent when started on day 4 or 5, but could
contribute to growth inhibition if it were started on day 1.
However, when used starting on day 4 or 5, in combination with
cisplatin, given at or near a maximum tolerated dose, squalamine
significantly and reproducibly improved tumor growth inhibition
over cisplatin alone in a dose-dependent fashion for both the H460
and Calu-6 cell lines.
d. Squalamine Use in Metastatic Lung Cancer
The murine Lewis lung adenocarcinoma was implanted subcutaneously
in the hind-leg of male C57BL/6 mice and allowed to grow for one
week. Groups of mice were then left untreated or treated with
either squalamine (20 mg/kg/day, s.c.), cyclophosphamide (125
mg/kg, i.p. on days 7, 9 and 11), cisplatin (10 mg/kg, i.p. on day
7), the combination of squalamine and cyclophosphamide, or the
combination of squalamine and cisplatin. On day 20, the animals
were sacrificed, and the mean number of lung metastases were
determined for each group. All treatments reduced the number of
metastases; however, the most effective treatments were the
combination of squalamine with either of the cytotoxic agents (FIG.
13).
II. THERAPEUTIC ADMINISTRATION AND COMPOSITIONS
The mode of administration of squalamine may be selected to suit
the particular therapeutic use. Modes of administration generally
include, but are not limited to, transdermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, inhalation,
intralymphatic, intralesional, and oral routes. The squalamine
compounds may be administered by any convenient route, for example,
by infusion or bolus injection, or by absorption through epithelial
or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal
mucosa, etc.), and it may be administered together with other
biologically active agents. Administration may be local or
systemic.
The present invention also provides pharmaceutical compositions
which include squalamine as an active ingredient. Such compositions
include a therapeutically effective amount of squalamine and a
pharmaceutically acceptable carrier or excipient. Examples of such
a carrier include, but are not limited to, saline, buffered saline,
dextrose, water, oil in water microemulsions such as Intralipid,
glycerol, and ethanol, and combinations thereof. The formulation of
the pharmaceutical composition should be selected to suit the mode
of administration.
The pharmaceutical composition, if desired, also may contain
effective amounts of wetting or emulsifying agents, or pH buffering
agents. The pharmaceutical composition may be in any suitable form,
such as a liquid solution, suspension, emulsion, tablet, pill,
capsule, sustained release formulation, or powder. The composition
also may be formulated as a suppository, with traditional binders
and carriers, such as triglycerides. Oral formulations may include
standard carriers, such as pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc.
Various delivery systems are known and may be used to administer a
therapeutic compound of the invention, e.g., encapsulation in
liposomes, microparticles, enteric coated systems, microcapsules,
and the like.
In one embodiment, the pharmaceutical composition is formulated in
accordance with routine procedures to provide a composition adapted
for intravenous administration to humans. Typically, compositions
for intravenous administration are solutions in 5% dextrose and
sterile water or Interlipid. Where necessary, the pharmaceutical
composition also may include a solubilizing agent and a local
anesthetic to ameliorate pain at the site of an injection.
Generally, the ingredients of the pharmaceutical composition are
supplied either separately or mixed together in unit dosage form,
for example, as a dry lyophilized powder or water-free concentrate
in a hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the pharmaceutical
composition is to be administered by infusion, it may be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water, dextrose, saline, or other pharmaceutically acceptable
carriers. Where the pharmaceutical composition is administered by
injection, an ampoule of sterile water or saline for injection may
be provided so that the ingredients may be mixed prior to
administration.
The amount of the therapeutic compound (i.e., active ingredient)
which will be effective in the treatment of a particular disorder
or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques
known to those skilled in the art. The precise dose to be employed
in the formulation also will depend on the route of administration
and the seriousness of the disease or disorder, and should be
decided according to the judgement of the practitioner and each
patient's circumstances. Effective therapeutical doses may be
estimated from extrapolations of dose-response curves derived from
in vitro or animal-model test systems.
Suitable dosages for intravenous administration are generally about
1 microgram to 40 milligrams of active compound per kilogram body
weight. Suitable dosage ranges for intranasal administration are
generally about 0.01 mg/kg body weight to 20 mg/kg body weight.
Suitable dosages for oral administration are generally about 500
micrograms to 800 milligrams per kilogram body weight, and
preferably about 1-200 mg/kg body weight. Suppositories generally
contain, as the active ingredient, 0.5 to 10% by weight of
squalamine. Oral formulations preferably contain 10% to 95% active
ingredient.
For use of squalamine as an antiangiogenic or cytotoxic agent or in
cancer therapies, exemplary dosages are from about 0.01 mg/kg body
weight to about 100 mg/kg body weight. Preferred dosages are from
0.1 to 40 mg/kg body weight.
The invention also may include a pharmaceutical pack or kit
including one or more containers filled with the pharmaceutical
compositions in accordance with the invention. Associated with such
containers may be a notice in the form prescribed by a government
agency regulating the manufacture, use or sale of pharmaceuticals
or biological products, which notice reflects approval by the
agency of manufacture, use or sale for human administration.
The conventional cytotoxic chemical compounds used in accordance
with the invention may be present in any suitable form known to
those skilled in the art. These chemical compounds also may be
administered by any suitable means also known to those skilled in
this art, such as orally, subcutaneously, intravenously,
intraperitoneally, intralymphaticly, and intramuscularly.
In describing the invention, applicants have stated certain
theories in an effort to disclose how and why the invention works
in the manner in which it works. These theories are set forth for
informational purposes only. Applicants are not to be bound to any
specific chemical or physical mechanisms or theories of
operation.
While the invention has been described in terms of various specific
preferred embodiments and specific examples, those skilled in the
art will recognize that various changes and modifications can be
madeithout departing from the spirit and scope of the invention, as
defined in the appended claims.
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